Triple-stranded DNA

A long-standing interest of my group has been the triple-stranded DNA structures, that may form in homopyrimidine•homopurine (Py•Pu) sequences, their possible biological significance, and real and potential practical use. Substantial amount of work has been done to understand how the high-energy triplex structure can be stabilized so that it may exist in less than exotic environments. The presence of divalent metal cations in cells (magnesium and zinc that support activities of many enzymes) prompted their investigation as possible triplex stabilizers. As a result, we proposed that coordination of transition metal cations to purine nucleotides in the third strand may cause polarization of the base charges, eventually leading to stronger hydrogen bonds with the complementary purine nucleotides in one strand of the target DNA duplex, thereby strengthening the triplex structure (Figure 1) [1]. Another source of triplex stability is related to stretches of cationic amino acid residues in proteins. In vitro experiments showed that cationic peptides, as models of protein domains, significantly stabilized the formation of intermolecular triplex between the linear DNA target and an appropriate third strand oligonucleotide, as well as supercoil-driven formation of intramolecular triplex (H-DNA) [3,5]. Conjugation of the triplex-forming oligonucleotide with cationic peptide stabilizer results in a significant acceleration of triplex formation. Several publications relevant to the biological significance of triplex formation describe H-DNA formation in the promoters of the Na,K-ATPase alpha2 [4] and c-myb genes [8], in the intron 21 of the PKD1 gene (Figure 3) [6], and within the long repeated (GAA)n•(TTC)n sequences [11], as well as strong inhibition of DNA polymerization on a homopurine template (Figure 2) [7]. In hope to make other people's lives easier, I reviewed different aspects of triplex DNA [2,10].

Publications

1. Potaman VN, Soyfer VN (1994) Divalent metal cations upon coordination to the N7 of purines differentially stabilize the PyPuPu DNA triplex due to unequal Hoogsteen-type hydrogen bond enhancement. J Biomol Struct Dyn 11: 1035-1040 (request a copy).
2. Soyfer VN, Potaman VN (1995) Triple-Helical Nucleic Acids. Springer. 360 pp.
3. Potaman VN, Sinden RR (1995) Stabilization of triple-helical nucleic acids by basic oligopeptides. Biochemistry 34: 14885-14892 (request a copy).
4. Potaman VN, Ussery DW, Sinden RR (1996) Formation of a combined H-DNA/open TATA box structure in the promoter sequence of the human Na,K-ATPase alpha2 gene. J Biol Chem 271: 13441-13447 (link to PDF).
5. Potaman VN, Sinden RR (1998) Stabilization of intramolecular triple/single-strand structure by cationic peptides. Biochemistry 37: 12952-12961 (request a copy).
6. Blaszak RT, Potaman V, Sinden RR, Bissler JJ (1999) DNA structural transitions within the PKD1 gene. Nucleic Acids Res. 27: 2610-2617 (link to PDF).
7. Potaman VN, Bissler JJ (1999) Overcoming a barrier for DNA polymerization in triplex-forming sequences. Nucleic Acids Res. 27: e5 (link to PDF).
8.Vigneswaran N, Thayaparan J, Knops J, Trent J, Potaman V, Miller DM, Zacharias W (2001) Intra- and intermolecular triplex DNA formation in the murine c-myb proto-oncogene promoter are inhibited by mithramycin. Biol Chem 382: 329-342 (request a copy).
9. Tiner WJ Sr, Potaman VN, Sinden RR, Lyubchenko YL (2001) The structure of intramolecular triplex DNA: atomic force microscopy study. J Mol Biol. 314: 353-357 (request a copy).
10. Potaman VN (2003) Applications of triple-stranded nucleic acid structures to DNA purification, detection and analysis. Expert Rev. Mol. Diagn. 3: 481-496 (request a copy).
11. Potaman VN, Oussatcheva EA, Lyubchenko YL, Shlyakhtenko LS, Bidichandani SI, Ashizawa T, Sinden RR (2004) Length-dependent structure formation in Friedreich ataxia (GAA)n•(TTC)n repeats at neutral pH. Nucleic Acids Res. 32: 1224-1231 (link to PDF).

Figure 1. Schematics of the proposed Hoogsteen-type hydrogen bond enhancement when divalent metal cations coordinate to the N7 position of the third strand purines [1].

Figure 2. Mechanism of polymerization arrest on a mirror-repeated purine template and its elimination. A. A 46-bp purine sequence from the PKD1 gene [6] and an oligonucleotide that hybridizes across the repeat center. B. The polymerization arrest may occur after a nascent pyrimidine strand reaches the center of the mirror repeat. The Watson-Crick duplex between the nascent pyrimidine strand and a half of the purine template serves as a target for binding via Hoogsteen hydrogen bonds of the other half of purine template still unused for DNA polymerization. Polymerase is inable to untangle the triplex and/or continue synthesis around the hairpin tip. C. Oligonucleotide hybridization across the mirror repeat center prevents purine strand folding into the hairpin. The progressing polymerase displaces the oligonucleotide and synthesizes a new strand on an unfolded template [7].

Figure 3. AFM image of intramolecular triple/single stranded DNA (H-DNA) in the (GAA)42•(TTC)42 repeats [11]. Similar images were obtained for H-DNA formed by the Py•Pu sequences from the PKD1 gene [9].